12 results about "Calcifediol" patented technology

The invention relates to the technical field of biology, in particular to a pichia pastoris recombinant strain for expressing non-specific peroxygenase CciUPO and application of the pichia pastoris recombinant strain. The pichia pastoris recombinant strain is obtained by the following steps: S1, synthesizing a sequence of a CciUPO original gene sequence optimized based on a yeast codon; s2, carrying out recombination with the EV signal peptide after the directed evolution; s3, connecting the recombined target gene with an expression vector to construct a recombinant plasmid; and S4, transforming the recombinant plasmid into pichia pastoris competent cells, and screening out a recombinant strain containing the CciUPO recombinant gene. The expression quantity of the CciUPO obtained through the operation is increased by 10 times compared with that of a wild type CciUPO, the CciUPO has strict catalytic regioselectivity, can catalyze vitamin D3 to synthesize calcifediol, the selectivity is as high as 95%, the catalytic conversion rate of toluene is 95%, the selectivity is 78%, and the CciUPO has a relatively high application prospect in industrial production.

The application belongs to the technical field of bio-chemical industry, and particularly relates to a P450 enzyme mutant and application of an engineering bacterium thereof in synthesis of calcifediol, the mutant is one or more point positions in an amino acid sequence based on a wild type P450 enzyme Vdh being mutated; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to GenBank accession number CP069288.1. Through systematic molecular dynamics simulation, conservation analysis and substrate binding pocket engineering on the Vdh enzyme, multiple key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I114R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, and the pure enzyme activity can reach 9.8 times of the wild type enzyme, greatly improving the conversion rate from vitamin D3 to calcifediol.

The invention belongs to the technical field of biological catalysis, and discloses a non-specific peroxygenase mutant, a preparation method and application thereof, and a preparation method of calcifediol. Compared with a wild type non-specific peroxygenase (comprising an amino acid fragment with a sequence as shown in SEQ ID NO: 1), the non-specific peroxygenase mutant provided by the invention has one or more of Y47L mutation, L83F mutation and V112E mutation. The non-specific peroxygenase mutant has excellent enzyme activity, has the advantages of high conversion rate, mild reaction conditions, low cost and the like when being applied to preparation of calcifediol, and has a good industrial application prospect.

The invention belongs to the technical field of biochemical engineering, and particularly relates to application of a P450 enzyme mutant and engineering bacteria thereof in synthesis of calcifediol, and the mutant is obtained by mutating one or more sites in an amino acid sequence based on a wild type P450 enzyme Vdh; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to the accession number of GenBank, namely CP069288.1. Through systematic molecular dynamics simulation, conservative analysis and substrate binding pocket engineering on the Vdh enzyme, a plurality of key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I144R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, the pure enzyme activity of the combined mutant can reach 9.8 times that of a wild type enzyme, and the conversion rate from vitamin D3 to calcifediol is greatly increased.

The invention discloses a calcifediol spray drying tower air inlet temperature control device and method, and relates to the technical field of calcifediol production equipment.The calcifediol spray drying tower air inlet temperature control device comprises an air inlet pipe, a plurality of heating pipes are arranged in the air inlet pipe at equal intervals, and a plurality of temperature sensors are fixedly arranged on the surface of the air inlet pipe; the temperature sensors are evenly distributed in the air inlet pipe in the circumferential direction and the axial direction of the air inlet pipe, and inductive probes of the temperature sensors penetrate through the air inlet pipe and extend into the air inlet pipe. The device has the beneficial effects that the plurality of temperature sensors which are uniformly distributed in the circumferential direction and the axial direction of the air inlet pipe and of which the sensing probes extend into the air inlet pipe can comprehensively detect the air temperature at different positions in the air inlet pipe, and compared with a single sensor, more accurate and comprehensive temperature data can be obtained, a reliable basis is provided for subsequent temperature regulation and control, and the device is suitable for popularization and application. The accuracy of inlet air temperature detection is effectively improved, and then the temperature control precision is improved.

The present invention discloses novel method for synthesizing vegan 25-OH cholesterol / Calcifediol from inexpensive crude phytosterol. According to the method, Phytosterols are reacted to form corresponding i-steroid through tosylation and methanolysis. i-steroid on reductive ozonolysis to C-22 alcohol and conversion via C-22 tosylate to C-22 iodide in good yield. Coupling of C-22 tosylate with Grignard reagent of 4-bromo-2-methyl-2-[(trimethylsilyl)oxy] butane followed by deprotection yielded 25-OH cholesterol. In a process variant, nickel mediated conjugate addition of C-22 iodide to an electron deficient alkene ethyl acrylate and treating corresponding ester with methyl magnesium bromide as means of installing the side chain of 25-OH cholesterol in high yield. Further bromination reaction of 25-OH cholesterol diacetate followed by dehydrobromination using TBAF yielded 25-OH 7-dehydrocholesterol. Further photo reaction of 25-OH 7-dehydrocholesterol in to previtamin D3 using high or medium pressure mercury lamp and subsequent thermal reaction of previtamin D3 to 25-OH vitamin D3(Calcifediol) in good yield.

The invention belongs to the technical field of biochemical engineering, and particularly relates to a P450 fusion protein, the structure of the fusion protein is VdhM3-Linker1-AciC-Linker2-AciB, VdhM3 is Vdh-M3 (I144R / N173M / Q310R), and VdhM3 is Vdh-M3 (I144R / N173M / Q310R); the GenBank login numbers of the coding genes of the AciC and the AciB are both AB221118.1, and the GenBank login numbers of the coding genes of the AciC and the AciB The amino acid sequence of the Linker2 is GSGSGH; and the Linker 1 is a flexible connector, a rigid connector or a flexible-rigid combined connector. By means of system screening, it is determined that the AciC / AciB redox partner system derived from acinetobacter has the optimal suitability with VdhM3 P450 enzyme, the catalytic activity is high, the selectivity of the target product calcifediol reaches up to 95%, generation of by-products is reduced, and the purification efficiency and yield of the product are improved.

The application discloses a method for extracting calcifediol from a crystallization mother liquor of a calcifediol crude product. The method uses a crystallization technique to extract the crystallization mother liquor of the calcifediol crude product obtained through a photochemical reaction for a second time to obtain a calcifediol crystal with a higher content, and then through a recrystallization process, a crystalline pure product meeting product quality standards is obtained. The specific method is as follows: after the solvent of the mother liquor after purification of the calcifediol crude product is evaporated, an oily concentrate is obtained, the oily concentrate is dissolved with an organic solvent, then activated carbon is added to decolorize at a certain temperature, filtration is performed, cooling crystallization is performed, filtration is performed, the solid is dissolved with an organic solvent for a second time, cooling crystallization is performed, filtration is performed, and drying is performed to obtain a finished product. The application has the advantages of simple process, short flow, high efficiency and high product purity.

The application belongs to the technical field of bio-chemical industry, and particularly relates to a P450 fusion protein, and a structure of the fusion protein is VdhM3-Linker1-AciC-Linker2-AciB, wherein VdhM3 is Vdh-M3 (I114R / N173M / Q310R); the GenBank accession numbers of the encoding genes of AciC and AciB are both AB221118.1; the amino acid sequence of Linker2 is GSGSGH; and Linker1 is a flexible linker, a rigid linker or a flexible-rigid combined linker. Through systematic screening, it is determined that the AciC / AciB oxidation-reduction partner system derived from Acinetobacter has the best adaptability with the VdhM3 P450 enzyme, the catalytic activity is high, and the selectivity of the target product calcifediol is as high as 95%, the generation of by-products is reduced, and the purification efficiency and yield of the product are improved.

Disclosed herein is an improved and efficient process for synthesis of vitamin D3 and its analogue Calcifediol from Ergosterol. Particularly, the present invention discloses the synthesis of key intermediate 3β-tert-Butyldimethylsilyloxy-22-hydroxy-23,24-bisnorchola-5,7-diene (5), and novel intermediate β-tert-Butyldimethylsilyloxy-22-iodo-23,24-bisnorchola-5,7-diene (9) by a simple and cost effective process. The industrially viable processes for preparation of said intermediate(s) results in providing provitamins with various side chains and the desired products in high yield.

The invention belongs to the technical field of biological catalysis, and particularly discloses a method for synthesizing calcifediol by using a photocatalytic device. According to the method, 7-dehydrocholesterol is used as a substrate, and 25-hydroxy-7-dehydrocholesterol is obtained through the steps of non-specific oxide enzyme catalysis, extraction, reduced pressure distillation and the like. Dissolving the 25-hydroxy-7-dehydrocholesterol in a polar solvent, a non-polar solvent or a mixed solvent system of the polar solvent and the non-polar solvent, adding an anti-oxidation reagent, and carrying out an illumination isomerization reaction; and under the protection of inert gas, carrying out thermal isomerization reaction on the reaction liquid after photochemical treatment, then carrying out low-temperature crystallization to obtain part of calcifediol crystals, collecting the filtrate after crystallization, and carrying out thermal isomerization reaction and low-temperature crystallization for multiple times to obtain the residual calcifediol crystals. The method has the advantages of simple operation steps, small usage amount of the organic solvent, high synthesis efficiency and cost saving, and is beneficial to large-scale industrial production of calcifediol.